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Minimal Tags for Rapid Dual-Color Live-Cell Labeling and Super-Resolution Microscopy

Authors

  • Dr. Ivana Nikić,

    1. Structural and Computational Biology Unit and Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg (Germany)
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  • Dr. Tilman Plass,

    1. Structural and Computational Biology Unit and Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg (Germany)
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  • Dr. Oliver Schraidt,

    1. Structural and Computational Biology Unit and Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg (Germany)
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  • Dr. Jędrzej Szymański,

    1. Structural and Computational Biology Unit and Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg (Germany)
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  • Dr. John A. G. Briggs,

    1. Structural and Computational Biology Unit and Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg (Germany)
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  • Priv.-Doz. Dr. Carsten Schultz,

    1. Structural and Computational Biology Unit and Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg (Germany)
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  • Dr. Edward A. Lemke

    Corresponding author
    1. Structural and Computational Biology Unit and Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg (Germany)
    • Structural and Computational Biology Unit and Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg (Germany)

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  • We thank S. Rizzoli for helpful discussions, N. Banterle, N. Davey, G. Estrada Girona, C. Koehler, S. Milles, S. Prinz, and S. Welsch for expert technical help, and H. G. Kraeusslich for the influenza plasmids. We thank V. VanDelinder and members of the Lemke group for proofreading this manuscript and for helpful discussions. The authors declare a conflict of interest, as some UAAs are covered by a patent application. This study was technically supported by the EMBL ALMF and PCF facilities. I.N. is an EMBO long-term fellow and T.P. a VCI fellow. O.S. is supported by a cofinanced Marie Curie/EIPOD fellowship. E.A.L. acknowledges funding by the Emmy Noether program and SPP1623 and CS from SPP1623 of the DFG.

Abstract

The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized organic dyes by the inverse-electron-demand Diels–Alder cycloaddition (SPIEDAC). Furthermore, we fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resolution microscopy.

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